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Corresponding author: Filip S. Šibul,
Sad Faculty of Sciences, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia
E-mail: filip.sibul@dh.uns.ac.rs, Phone: +381 21 485 2765 Fax: +381 21 455 662
1
Abstract
Non-optimized extraction conditions can lead to losses, degradation and modification of the
biomolecules. In this paper, the influence of different solvent mixtures, solvent amounts,
temperature, extraction time, and procedures for defatting on yield and profile of various
classes of secondary metabolites was investigated. Rumex alpinus was used for the extraction
and phenolic acids, Anthriscus sylvestris for lignans and coumarins, alkaloids were extracted
from Lupinus albus and sesquiterpene lactones from Artemisia absinthium. Extraction
solvent for all of the examined compounds is 80 % methanol, mixed in ratio 13 : 1 with plant
material. Maceration should last for six hours, repeated four times with fresh solvent.
Defatting of the extracts does not lead to significant losses of the compounds of interest. It is
acceptable to use extraction and evaporation temperature of 60 ºC, while the extracts should
2
INTRODUCTION
For centuries, Chinese traditional medicine has been using nature as a source of
compounds which can be used in treatment of various diseases. Today, major attention of
plants [1].
defense from herbivores, bacteria, viruses, fungi and other organisms. By targeting different
receptors and enzymes in predators and parasites, they have developed different mechanisms
while isoflavonoids have phytoestrogen abilities, due to their structural similarity with
components extracted from Rumex alpinus (Polygonaceae), are reported to have anti-
inflammatory, antifungal and antibacterial activity. Isoflavonoids extracted from Glycine max
(Fabaceae) are phytoestrogens, while flavonoids and phenolic acids from Chaerophyllum
Anthriscus sylvestris (Apiaceae) is rich in lignans and coumarins, known for their anticancer,
responsible for their bitter taste, as well as their antimicrobial activity [2,4].
Extraction of the active principles is an essential step in evaluation of their bioactivity and
result in invalid conclusions about the chemical composition and the amount of secondary
maximizing yields of the compounds of interest, while minimizing the extraction of unwanted
compounds. In this paper, we investigated the influence of different solvent mixtures, solvent
amounts, temperature, extraction time, techniques, and procedures for fatty acid and
chlorophyll removal on yield and profile of various classes of secondary metabolites from the
selected plant species, during maceration. Extraction efficiency was evaluated by using
compound classes.
EXPERIMENTAL
Chemicals and reagents. HPLC gradient grade methanol, p.a. ethanol and methanol were
purchased from J. T. Baker (Deventer, The Netherlands), and p.a. DMSO from Merck
(Darmstadt, Germany).
Plant materials. The plant material used for extraction and analysis was collected from
different locations in Serbia – Chaerophyllum bulbosum (CB) from Vlasinsko Jezero lake and
Anthriscus sylvestris (AS) from Fruška Gora mountain in 2009, Artemisia absinthium (AA)
from Stara Planina mountain in 2011, Rumex alpinus (RA) from Kopaonik mountain in 2012,
while Glycine max (GM) and Lupinus albus (LA) were collected from Rimski Šančevi,
agrarian area in the Panonian basin, in 2013. Voucher specimens were prepared, identified
and deposited at the Herbarium of the Department of Biology and Ecology (BUNS
4
The selected plant species were used for extraction of different secondary metabolites present
in their aerial parts, as determined by our preliminary studies. Herb of Rumex alpinus (monk's
rhubarb, Alpine dock) was used for extraction of anthraquinones, Glycine max (soy) for
phenolic acids. Aerial parts of Anthriscus sylvestris (wild chervil) were used as a source of
lignans, Lupinus albus (white lupin) contains alkaloids, while the herb of Artemisia
Extracts Preparation
Plant material was air-dried at room temperature, and aerial parts were separated from roots
and powdered afterwards. All extracts were prepared by maceration. During the extraction
process, aerial parts were constantly shaken at three different temperatures, during various
periods of time, and using several solvent mixtures (as described in the following
subsections). The composition of the extraction medium, as well as the volume of the solvent,
extraction time and temperature, were optimized during the experiment to obtain the highest
depending on the compound class). Plant material was removed by filtration, raw extracts
were evaporated under the reduced pressure and reconstituted in DMSO to the final
concentration of 50 mg/mL.
Solvent mixture selection. For the purpose of testing the influence of different solvent
mixtures on secondary metabolites extraction, 100 mg portions of each dried plant material
were mixed in 1.8 mL HPLC vial with 1 mL of the following solvents: water, 60% methanol,
80% methanol, methanol, 60% ethanol, 80% ethanol, and ethanol. The mixtures were shaken
for 60 min at room temperature on vortex mixer, and filtered through millipore filters (0.45
compounds were identified by comparing their molecular weights, mass spectra and UV/Vis
5
spectra with the literature (refer to the Section 'Identification of compounds present in plant
extracts'). Further on, their abundance profiles obtained from extraction by different solvents
were compared. The goal was to achieve the maximum possible yield of a wide range of
biomolecules identified in the selected plants. Afterwards, for each of the extracts, LC-
MS/MS or LC-DAD method was created to monitor the yield of the compounds of interest.
Optimal extraction volume selection. To find the optimal extraction medium volume, 1 mL
of the optimal solvent was mixed with 50 mg (solvent mixture to plant material ratio of 1:20),
75 mg (1:13.3), 100 mg (1:10), and 125 mg (1:8) of the dry plant material. After shaking for
60 min on vortex mixer and filtering through millipore filters, all mixtures were diluted to the
concentration of 50 mg/mL, for analysis with LC-ESI-MS/MS. Plots showing peak areas
Extraction kinetics. For the purpose of extraction kinetics evaluation, 5 g of the drug was
mixed with the optimal volume of extraction solvent in 250 mL Erlenmeyer flask, closed to
avoid evaporation of solvents, and shaken at room temperature for 73 h. Sample aliquots of
adding the extraction medium, filtered and analyzed by LC-DAD-ESI-MS technique. The
Multiple extractions. 500 mg of the plant material was repeatedly extracted for 5 times with
an optimal solvent volume. Each of the five extractions was performed by shaking the mixture
for 1 hour at room temperature, filtering out the extract using vacuum, and re-extracting the
remaining plant material with the same amount of fresh extraction medium. 500 µL of each
cycle was calculated, assuming approximately 100 % recovery after the fifth extraction, and
6
Optimal temperature selection. Optimization of extraction temperature was performed by
shaking 100 mg of the plant material with the optimal extraction medium for 60 min at room
temperature (25 ºC), and at 60 ºC and 100 ºC in water bath. After filtering, the extracts were
Evaporation under reduced pressure. To increase the throughput, the maximum acceptable
evaporation temperature was assayed. Extracts prepared in the experiment evaluating the
extraction kinetics (after 73 h) were evaporated under the reduced pressure, using rotary
evaporator with bath temperature set on 30 ºC, 45 ºC and 60 ºC. Dry residues were
Unwanted compounds removal. Examination of losses during removal of fats and pigments
vacuo. Dry residue was suspended in 1 mL of warm water (~60 ºC), additional 1 mL of water
was then added to wash the evaporation flask, and the water solutions were mixed. To 2 mL
of this solution, 2 mL of hexane was added, the content was shaken on vortex mixer and then
frozen using dry ice. Hexane layer was decanted from the frozen aqueous layer, and then new
portion of hexane (2 mL) was added to the thawed aqueous layer. This procedure was
repeated for five times, resulting in 2 mL of aqueous phase and 10 mL of pooled hexane
extracts. Both phases were evaporated separately in vacuo, dry residues were reconstituted to
The obtained chromatograms were compared to evaluate the compound distribution between
conditions were stored for 6 months under different conditions: at room temperature exposed
to sunlight, at room temperature in the darkness, at 4 ºC in the darkness, and at -20 ºC in the
darkness. In addition, one portion was diluted in typical HPLC mobile phase (0.05 % aqueous
formic acid : methanol = 1 : 1) and kept at 4 ºC in the darkness. After six months, LC-DAD-
ESI-MS/MS analysis of all samples was performed, and profiles of the compounds of interest
were compared for the purpose of evaluating possible losses and degradation.
LC-ESI-MS analysis
Extracts were diluted with mobile phase solvents A (0.05 % aqueous formic acid) and B
(methanol), premixed in 1:1 ratio, to obtain a final concentration of 50 mg/mL. Extracts were
analysed using Agilent Technologies series 1200 HPLC instrument coupled with Agilent
Technologies 6410A Triple Quad tandem mass spectrometer with electrospray ion source,
(ver. B.03.01). Five microlitres were injected into the system, and compounds were separated
on Zorbax Eclipse XDB-C18 (50 mm × 4.6 mm, 1.8 µm) rapid resolution column held at 50
ºC. Mobile phase was delivered at flow rate of 1 mL/min in gradient mode (0 min 30% B, 6
min 70% B, 9 min 100% B, 12 min 100 % B, re-equilibration time 3 min). Eluted components
MS(–) experiments. For detection of flavonoids and phenolic acids from Chaerophyllum
bulbosum and isoflavonoids from Glycine max, MS-ESI ion source parameters were as
follows: nebulization gas (N 2 ) pressure 50 psi, drying gas (N 2 ) flow 9 L/min and temperature
350 ºC, capillary voltage 4 kV, negative polarity (NI). For general screening, data were
acquired in MS2Scan mode, using m/z range from 120 to 1000 and fragmentor voltage of 100
V. For targeting compounds of interest, MS2SIM mode was used, with specific m/z values:
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643, 601, 515, and 353 for acetyl-malonyl-dicaffeoylquinic acid (AcMalC 2 QA), malonyl-
dicaffeoylquinic acid (MalC2 QA), dicaffeoylquinic acid (C 2 QA) and caffeoylquinic acid
(CQA); 489, 463, 447, 431, and 285 for luteolin/kaempferol acetylhexosides (Lut/Kaempf-
Chaerophyllum bulbosum. In Glycine max, m/z 269 and 253 were targeted for genistein and
daidzein, respectively.
MS(+) experiments. Ion source parameters for screening of alkaloids from Lupinus albus
were identical to those described in section 2.4.1, except for the use of positive polarity (PI).
For targeting the compounds of interest, MS2SIM in positive mode was used, with specific
m/z values: 347, 265, and 249 for angeloyloxy/tigloyloxy-lupanin, hydroxylupanin and
lupanin, respectively.
The above said parameters were not suitable for analysis of sesquiterpene lactones from
Artemisia absinthium, due to inefficient ionization by ESI source. Therefore, the analysis of
this plant's extracts was performed using an instrument with multimode ion source.
Instrument configuration was identical to the one above, with a difference in MMI source
parameters: nebulization gas (N 2 ) pressure 50 psi, vaporizer temperature (APCI heater) 200
ºC, drying gas (N 2 ) flow 5 L/min and temperature 325 ºC, capillary voltage 2.5 kV, positive
polarity (PI). In MS2SIM mode, compounds of interest were targeted on m/z values: 513, 497,
UV/Vis Detection. For analysis of lignans present in Anthriscus sylvestris herb extract, UV
signals at 280 nm and 330 nm were monitored, with bandwidth of 16 nm. Anthraquinones
from extract of Rumex alpinus were monitored in visible area at 430 nm, with bandwidth of
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16 nm. For both classes, UV/VIS was found to be more practical than MS in detecting all
Additionally, to confirm the identity of peaks, continuous spectra were obtained in range from
Chaerophyllum bulbosum extracts were identified by MS1 and MS2 analysis in negative
ionization (NI) mode, according to the rules set by Clifford et al., 2003 [5]. Examination of
the total ion chromatograms (TIC) resulted in four signals: m/z 353 for caffeolyquinic acids
(CQA), m/z 515 for dicaffeoylquinic acids (C2 QA), m/z 601 for malonyl-dicaffeoylquinic
acids (MalC2 QA), and m/z 643 for acetyl-malonyl-dicaffeoylquinic acids (AcMalC 2 QA).
CQAs were identified at retention times (tR) of 0.7 min, 0.8 min and 1.4 min. C 2 QAs eluted at
tR 1.7 min, 1.8 min, 1.9 min and 2.35 min, MalC 2 QAs at 1.6 min and 1.85 min, and
Flavonoids were also identified in Chaerophyllum bulbosum extracts after MS1 and MS2
analysis in negative ionization (NI) mode, according to the rules set by Cuyckens et al., 2000
[6]. Examination of the total ion chromatogram (TIC) resulted in five signals: m/z 285 for
luteolin and kaempferol (Lut/Kaempf), m/z 431 for apigenin hexosides and
kaempferol hexosides (Lut/Kaempf-Hex), 463 for quercetine hexosides (Quer-Hex), and m/z
489 for luteolin and kaempferol acetyl hexoside (Lut/Kaempf-AcHex). The only peak in the
EIC signal at m/z 285 was identified as luteolin (Lut) by comparison of the retention time (tR
10
= 3.8 min) with the reference standard. The EIC signal at m/z 431 contained two major peaks.
comparison with the reference standard. The second peak, with the retention time of 3.32 min,
weight and characteristic UV spectra. Two compounds were detected in EIC chromatogram at
m/z 447. By comparing the retention times with the reference standards, luteolin-7-O-
min) were identified. The signal at m/z 463 exhibited one major and one minor peak, both
corresponding to quercetin hexosides. By comparing the retention times with the reference
standards, it was confirmed that the first peak at tR 2.04 min was quercetin-3-O-galactoside
(isoquercitrin). Finally, Lut/Kaempf-AcHexs were identified at tR 2.4 min, 2.74 min and 2.9
min.
Isoflavonoids from Glycine max. Isoflavonoids in Glycine max extracts were identified after
MS2Scan analysis in negative ionization (NI) mode. Examination of the total ion
chromatogram (TIC) and the extracted ion chromatograms (EIC) resulted in two signals: m/z
253 for daidzein (Da) and m/z 269 for genistein (Gen). These compounds represent the two
major isoflavonoids present in soy, and were identified by their molecular masses, as well as
by comparing their MS2 and UV spectra with the reference data [7].
Alkaloids from Lupinus albus. Alkaloids in Lupinus albus extracts were detected by MS1
and MS2 analysis in positive ionization (PI) mode. Spectral data from the literature helped in
identification of the major compounds present in this plant [8,9]. Peak at 0.57 min belongs to
lupanin (m/z 249). Overlapping peaks with tR 0.54 and 0.69 min are identified as two isomeric
hydroxylupanins (m/z 265). Peak at 1.17 min corresponds to one of the two isomeric
present in Artemisia absinthium was performed using APCI MS in MS2SIM mode. The
compounds of interest were identified by comparison with the literature spectral data [8,10].
According to the data, peak at 4.9 min represents artabsin (m/z 249), anabsin eluted at tR 5.25
min (m/z 513), and peak at 6.93 min is absinthin (m/z 497).
Lignans from Anthriscus sylvestris. The lignans dominant in Anthriscus sylvestris extracts
were identified by using the literature data about this plant's chemical composition, including
UV spectra and retention information [11,12], as well as the fragmentation rules set by Wong
et al., 2000 [13]. Peaks at 4.47 min, 5.47 min, and 5.65 min have UV spectra characteristic for
and lack of maxima above 300 nm. Based on MS 2 data, these compounds were identified as
min and 6.49 min have absorbance maximum between 320 nm and 330 nm, and have been
peaks were detected, with retention and UV spectra similar to those of the compounds
identified.
were identified by their characteristic UV/Vis spectra, with a prominent absorbtion maximum
at 430 nm and a series of maxima under 300 nm. Compounds eluting between 4.5 min and 5.5
min were identified according to their molecular masses and retention times as anthraquinone
glycosides, while the compounds eluting later (from 7.9 min to 9 min) were identified as
anthraquinone aglycones.
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Extraction conditions optimization
donating and proton-accepting abilities, extract different classes of compounds from plant
material with a varying yield. With a goal of maximizing the yield of a wide range of
abundance profiles of the selected compounds, obtained by analysis after the extraction by
different solvent mixtures. According to literature data, optimal extraction mediums for most
of the plant compounds are methanol and ethanol, mixed with water in different ratio [14,15].
For all of the identified chlorogenic acids, 60 % and 80 % methanol were found to be the most
yield for dicaffeoylquinic acids and their malonyl derivatives, but lower for caffeoylquinic
and acetyl-malonyl-dicaffeoylquinic acids. Furthermore, it was noticed that water and neat
ethanol are not suitable for chlorogenic acids extraction, due to very low yields.
Extraction profiles for flavonoids from Chaerophyllum bulbosum depend on the structure.
Efficiency of glycosides extraction increased with methanol content, while for the ethanol-
based solvents it peaked at 80 % ethanol. Generally, the pure methanol was found to be the
most efficient, followed by 80 % methanol. As for lutheolin and its acetylhexosides, the
pattern was reversed, showing that 60 % and 80 % methanol provide higher yields than pure
methanol. Yields of flavonoids, obtained using water extraction, were very low. Surprisingly,
isoflavonoid aglycones daidzein and genistein were the most efficiently extracted with
solvents of intermediate polarity, while neat water and alcohols provided lower yields. The
effect of the solvent on the investigated sesquiterpene lactones extraction from Artemisia
absinthium was less pronounced. While the extraction efficiency of water was low, 60–100 %
alcoholic solvents provided high yields. Similarly, the most suitable extraction solvents for
ethanol were found to be inefficient. Polar solvents – water, 60–80 % methanol and 60 %
ethanol – were found to be the most efficient for extraction of the selected alkaloids (lupanin,
hydroxylupanin and angeloyl/tigloyloxylupanin) from Lupinus albus, while pure ethanol gave
very low yields. It should be noted that a pH adjustment of the sample may be needed for
compounds, water was found to be the unsatisfactory solvent. To obtain the maximum yield
extraction of the majority of compounds present in plants used in this experiment, with 60 %
ethanol as a viable alternative. This is in accordance with the literature data [14,15].
Extraction efficiency of different solvent mixtures towards various plant pigments, such as
chlorophylls and carotenes, was also evaluated. The pigments were identified by their UV/Vis
spectra, and their content was estimated from the total signal on λ=400–700 nm in late-eluting
region of a chromatogram. Pigments can interfere with the analysis of extracts, especially
biological activity. Therefore, the optimal sample preparation procedure should minimize co-
extraction of pigments with the compounds of interest. It was found that the selected optimal
extraction solvent, 80 % methanol, also extracts high amounts of pigments (although less than
60 % methanol, pure methanol and 80 % ethanol), therefore the extract purification may be
necessary. It was also observed that 80 % methanol extracts the highest amount of fatty acids
that can interfere with anthraquinones determination due to similar elution and isobaric
molecular weights, although these problems can be alleviated by using MS/MS or UV/VIS
detection.
14
Optimal extraction medium volume. Optimal extraction medium volume represents the
ratio of dry plant material weight to volume of the extraction solvent. Volume optimization
leads to use of the minimum amount of the extraction medium needed for a high yield of the
extracted compounds, thus saving large amounts of toxic and expensive solvents.
Furthermore, evaporating smaller amounts of solvent increases the extraction throughput and
reduces energy expenditure. As expected, the results show that the extraction yield
dramatically increases with the increase of solvent-to-drug ratio, due to increase of the
concentration gradient needed for efficient extraction. To obtain a high yield in one-step
extraction, the ratio of at least 20:1 should be used (Fig. 1). Since this results in a very high
solvent usage, a lower ratio of approx. 13:1, which provides approximately twice the yield of
commonly used 8:1 extraction for majority of the compounds investigated, was selected for
the following experiments. This result is in correlation with the recommended ratio of
Fig. 1.
Optimal extraction time. Extraction yield increases with time until the equilibrium is
reached. Prolonging the extraction after this point has little sense, since it leads to longer
experiment time, and also increases the chance of artifact formation through hydrolysis,
The obtained results show that concentrations of all compounds grow rapidly during the first
two hours of extraction, reaching the estimated yields of 70–90 %. Afterwards, the yield rises
slowly due to solvent saturation by the compounds present in plant material, to reach
15
extraction provides a satisfactory yield (approx. 84–93 %) within a reasonable amount of
Fig. 2.
repeated several times with a fresh portion of an extracting solvent. However, multistep
extraction leads to increased solvent usage, prolonged procedure, and a larger volume of
extract that needs to be evaporated. Thus, it is essential to optimize the number of solvent
Results show that the yield of one extraction step is between 42 % and 68 %, depending on
the compound class. The average cumulative yield increases at an expected rate in subsequent
steps, reaching 66–88 %, 84–94 % and 95–97 % in the second, third and fourth step.
Therefore, at least four consecutive extractions are required for obtaining acceptable yields
(Fig. 3). Considering the previous results, which showed that most of the components are
extracted after 6 h, we concluded that the optimal extraction should be performed 4 times for
90 min in order to get the maximum extraction yield within a reasonable time.
Fig. 3.
Extraction temperature. With increasing the temperature, solvent viscosity decreases, which
enhances a solvent penetration into plant material and increases the rate of extraction process.
thermolabile compounds and evaporation of the volatile ones. Extraction temperature is thus
16
optimized to provide a maximum yield of the compounds of interest, without their further
degradation.
For extraction of lignans from Anthriscus sylvestris, that are non-polar due to permethylation,
higher temperatures (100 ºC) were found to be beneficial, with yield at 100 °C about 33 %
higher than the one at 25 °C. This can be explained by increased solubility due to a decrease
of polarity of water in the extraction solvent [16]. The profile of the dominant lignans was
A very similar yield increase was observed for flavonoid aglycones and non-conjugated
while an equal number of peaks of the corresponding acetylhexosides increased. This was
expected, since it is well-known that malonyl esters undergo decarboxylation into acetates at
Similar effects were observed for chlorogenic acids from Chaerophyllum bulbosum. The
increase of temperature from 25°C to 60°C provides only a slight increase in yield of these
levels, and an increase in content of C 2 QA, AcC2 QA and AcMalC 2 QA (and CQA, to a lesser
extent). This indicates that, at elevated temperatures, MalC 2 QA undergoes hydrolysis (into
C2 QA and CQA) and malonyl moiety decomposition (into AcC 2 QA). The conversion into
AcMalC2 QA has not been previously observed, to the best of our knowledge.
Isoflavonoids from Glycine max, despite their structural similarity to other flavonoid
aglycones, behaved differently. While genistein yield changed only slightly, losses of
daidzein were observed at 60 °C and, especially, at 100 °C. The results are in agreement with
17
the previous findings demonstrating thermal lability of isoflavonoids. Studies conducted by
Ungar et al. indicated that daidzein is fairly stable while genistein undergoes decomposition,
as opposed to our findings. This discrepancy may be attributed to a difference in test systems,
30 %. Higher temperatures (100 °C) have no further effect on glycosides levels, while
aglycones yield drops. It should be noted that glycosides profile changes at 100 °C, indicating
possible interconversion.
Yield of alkaloids from Lupinus albus exhibited only a small change with a temperature
increase from 25 °C to 60 °C, while heating to 100 °C resulted in a more pronounced yield
drop. In the case of esterified hydroxylupanin, the highest yield was observed at 60 °C (Fig.
4).
Based on the results obtained, elevated temperature of 60 °C appears to be relatively safe, not
resulting in degradation of majority of the investigated compounds (even malonyl esters, that
are commonly claimed to degrade at above 35 °C), while increasing the yield of some
compounds. It can also be expected that the extraction equilibrium is reached faster due to a
avoided (except when specifically studying decoctions) since it leads to losses in several
compounds classes.
Fig. 4.
usually by using rotary evaporator under reduced pressure and below the solvent boiling
point. While elevated temperature enhances the process, it may lead to chemical modification,
18
degradation, or evaporation of the compounds of interest. Thus, it is necessary to find the
Results show that for the majority of the investigated compound classes there are no
preparation is needed. The notable exceptions are isoflavonoids daidzein and genistein, which
exhibit a sharp drop in concentration when evaporated at 60 °C. These compounds have
previously been reported to react with proteins at elevated temperatures, which decreases their
Losses during the defatting process. Extraction of the compounds of interest is usually
triacylglycerols and phospholipids). Since these compounds can interfere with compounds of
interest in further analyses, they should be removed. Removal of the lipophilic components is
performed by liquid-liquid extraction using non-polar solvents like hexane or petroleum ether.
However, some compounds of interest can also transfer to organic phase, resulting in a
Results show that defatting does not influence the yield of phenolic acids, flavonoids,
isoflavonoids and anthraquinone glycosides, due to their polar nature. Average losses were
about 0.8 %, and did not exceed 2.2 %. Transfer to hexane phase was more pronounced with
lupin alkaloids, reaching up to 8.5 % for hydroxylupanin. On the other hand, non-polar
compounds like sesquiterpene lactones from Artemisia absinthium and especially lignans
from Anthriscus sylvestris are lost to a significant extent during the defatting process (76 %
for lignanes and 29–57 % for sesquiterpenes). The situation is more complex with
19
anthraquinone aglycones. While apparent total loss is about 22 %, individual compounds
differ in their partitioning behavior, some remaining in water while others predominantly
Consequently, while there are no major losses of polar compounds of interest during the
Storage conditions. Often, it is not possible to analyze the extracts immediately after
extraction, which leads to the need for their storage for a prolonged period. During the
storage, partial or complete decomposition of compounds can take place, depending on the
Results show that after a period of six months, CQA, C 2 QA, MalC2 QA and AcMalC 2 QA are
almost completely degraded at room temperature in the presence of light, with their content
dropping to 0.1–13 % of the one observed in samples stored at –20 °C. While the abundance
of free quinic acid was slightly elevated, it was not possible to identify any other degradation
products, implying that chlorogenic acids decomposed mainly to low molecular weight
compounds (below used m/z threshold of 120). Caffeoyilquinic acid was stable at room
temperature in the dark. Under the same conditions, malonylated chlorogenic acids –
MalC2 QA and AcMalC 2 QA – decomposed almost completely, but this time clearly giving
rise to mono- and dicaffeoylquinic acids as the main products. At 4 °C, only MalC 2 QA
acids, samples should be kept in dark at –20 °C. The solvent also influences the stability of
chlorogenic acids – the sample kept at 4 °C in solvent containing 0.01 % formic acid and
temperature while being exposed to sunlight. The behaviour of flavonoid glycosides in the
absence of light depended on the structure. Levels of malonylhexosides decreased 1.6 – 2.6
during storage at 4 °C, while decomposition at room temperature was practically complete,
independent of insolation. Levels of acetylhexosides also dropped when exposed to light and
Finally, levels of hexosides increased with the temperature in the absence of illumination,
stored at –20°C, protected from light. Due to lower solubility of flavonoid aglycones in water,
a less polar solvent (e.g. methanol) should be present in sufficient amount (e.g. 80 %),
otherwise precipitation will occur, resulting in losses during filtration prior to HPLC analysis.
Genistein decomposed to a great extent in samples kept at room temperature (especially under
insolation), while daidzein was stable under all examined conditions. In samples diluted in
mobile phase and kept at 4 ºC, both of the examined isoflavonoids partially precipitated and
The obtained results show that light causes only partial degradation of lignans, less
pronounced than with chlorogenic acids and flavonoids. However, due to low polarity,
aqueous-alcoholic mixtures with high alcohol content should be used for storage if losses due
Anthraquinones from Rumex alpinus are known to be photosensitizers. Their aglycones were
21
light, while glycosides seem to be more resistant (only about 15 % was lost, when compared
hydroxylupanin content, points out their gradual hydrolysis at room temperature in the
presence of light. In the non-illuminated sample stored at room temperature, as well as in the
sample stored in acidic solvent (premixed mobile phase), the content of esters dropped to a
similar level, but without the accompanying rise in free alcohol, indicating different
degradation mechanism.
Based on the results obtained, it can be concluded that, due to instability of the majority of the
compounds investigated, samples should be stored in the freezer (–20 °C) and protected from
light.
CONCLUSION
The proposed optimized method is simple, and can be used for extraction of a wide range of
extraction yield for all examined compounds, mixed in ratio 13 : 1 with dry plant material.
Six-hour maceration, repeated for four times with fresh extraction solvent, provides a
provides highly efficient extraction. Since it does not lead to compound degradation, it can be
also used for crude extract evaporation. Defatting of extracts with hexane does not lead to
major losses of compounds of interest. Thus, it can be used for removal of fats and pigments.
Finally, results show that prepared extracts should be stored away from sunlight, on -20 °C.
22
Acknowledgments. This article was financially supported by a research grant from the
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25
Izvod
(Naučni rad)
vezanim za hemijski sastav biljke. Usled toga, cilj ovog rada bio je ispitivanje uticaja
Ključne reči: ekstrakcija, sekundarni metaboliti, biljni polifenoli, flavonoidi, fenolne kiseline
27
Figure captions:
Fig. 2. Peak areas of compounds extracted during different periods of time, cumulatively
Fig. 3. Peak areas of compounds extracted with different number of solvent portions
28
Figure 1.
Figure 2.
29
Figure 3.
Figure 4.
30